The Role of Mesenchymal Stem Cells and Exosomes in Tumor Development and Targeted Antitumor Therapies

Mesenchymal stem cells (MSCs) can be isolated from various tissues in adults and differentiated into cells of the osteoblasts, adipocytes, chondrocytes, and myocytes. Recruitments of MSCs towards tumors have a crucial contribution to tumor development. However, the role of MSCs in the tumor microenvironment is uncertain. In addition, due to its tropism to the tumor and low immunogenic properties, more and more pieces of evidence indicate that MSCs may be an ideal carrier for antitumor biologics such as cytokines, chemotherapeutic agents, and oncolytic viruses. Here, we review the existing knowledge on the anti- and protumorigenic effect of MSCs and their extracellular vesicles and exosomes, the role of MSCs, and their extracellular vesicles and exosomes as antitumor vectors.


The Antitumorigenic Activity of MSCs
MSCs are considered to be antitumor factors in hematological tumor cell lines, including Jurkat leukemia [19], human erythroid leukemia [20], Burkitt's lymphoma [21], non-Hodgkin's lymphoma [22], and T-cell lymphoma [23], and solid tumor cell lines such as breast cancer [24,25], hepatocellular carcinoma (HCC) [26,27], prostate cancer [25,28], melanoma [29], neck squamous cell carcinoma [30], Kaposi's sarcoma [31], pancreatic tumors [31], and multiple myeloma [32] (showed in Table 1). The mechanism of the antitumor effect of MSCs is multifaceted. Lin et al. showed that the growth of lymphoma cells was inhibited by the molecules secreted by umbilical cord MSCs (UC-MSCs) via the oxidative stress pathway by alteration of antioxidant enzymes [21]. In non-Hodgkin's lymphoma, MSCs induce endothelial cell migration in the Transwell test but promotes endothelial cell apoptosis in direct MSC/endothelial cell cocultures. The cytotoxic activity of MSC requires MSC/ endothelial cell contact [22]. The MSCs suppress tumor growth in vivo by inhibiting Akt activity in Kaposi's sarcoma [2]. Type I interferon is expressed in high-density cultured adipose tissue MSCs (AT-MSCs). AT-MSCs and their conditioned medium inhibit the growth of breast cancer MCF-7 cells in vitro [25]. Paracrine factors of human fetal MSCs inhibit liver cancer growth by reducing the activation of IGF-1R/PI3K/Akt signaling [26]. AT-MSCs can effectively inhibit the proliferation and division of HCC cells and induce HCC cell death by downregulating the Akt signaling pathway [27].UC-MSCs inhibit the proliferation of prostate cancer PC-3 cells by activating JNK and downregulating PI3K/AKT signals under coculture conditions [28]. Via the oxidative stress pathway by alteration of antioxidant enzymes [21] Non-Hodgkin's lymphoma

BM-MSC
In vitro and in vivo Induce endothelial cell migration in the Transwell assay, promote endothelial cell apoptosis in direct MSC/endothelial cell cocultures.

The Protumorigenic Activity of MSCs
However, studies are arguing for the protumorigenic role of MSCs on tumors, including tumor growth, angiogenesis, metastasis, and resistance to drugs [14]. The mechanism of supporting tumor vasculature of MSCs remains controversial. The biologically active factors secreted by MSCs contribute to the angiogenesis of tumors. Tumor-residing MSCs secreted high levels of VEGF in pancreatic carcinoma, which stimulates angiogenesis and increases microvessel density in the tumor [34]. The secretion of interleukin-6 (IL-6) from MSCs increases the secretion of endothelin-1 (ET-1) in colorectal cancer cells, which activates Akt and ERK in endothelial cells, thus enhancing their capacities for vessel formation [35]. Even more, the differentiation of MSCs into endothelial cells is a direct contribution to blood vessel formation. To examine the differentiation of MSCs into endothelial cells, the MSCs were cultured for several weeks in an endothelial cell culture medium containing VEGF. And the results showed that the expression of typical endothelial cell markers could be detected only in very few MSCs [34]. However, another study showed that melanoma cells educated MSCs to create vascular-like structures in vitro, thereby supporting tumor vasculature [36].
MSCs can facilitate tumor cell migration and invasion, EMT, and the formation of secondary metastatic lesions by a vast array of growth factors, cytokines, and chemokines derived from MSCs. MSC-derived C-C and CXC type chemokines, extracellular matrix modulating factors such as lysyl oxidase, and growth factors such as TGFβ, FGF, HGF, and EGF critically contribute to metastasis [37].
MSCs can promote tumor resistance to chemotherapy. IL-6, IL-7, IL-8, EGF, and IGF secreted from BM-MSCs induce chemoresistance to paclitaxel in head and neck carcinoma [38]. MSCs activated by platinum-based chemotherapy release two unique fatty acids inducing resistance to multiple types of chemotherapy [39]. Moreover, the clinical benefit of chemotherapy can be enhanced by blocking the release of these fatty acids from MSCs. Timaner et al. thought that MSCs could transform into cancer stem cells (CSCs) to support drug resistance [37].
As described above, some reports claim that MSCs can promote tumor progression, while others report that MSCs have antitumor effects. The observed differences may be due to the inherent biological differences in the source of MSCs, the heterogeneity of MSCs, the interaction of MSCs and their secreted factors with the surrounding microenvironment, and other parameters, such as the dose and time of MSCs administration/analysis, as well as the optimal culture condition [22,40]. The same amount of human amniotic membrane protein extract has different degrees of antipromoting or mitogenic effects on different tumor cells, indicating that the effect of MSCs may depend on the type of main receptors expressed on specific tumor cells [41]. This is why MSCs derived from different sources and acting in different tumor environments have tumor-promoting or antitumor behaviors.

MSCs as Antitumor Vectors
More and more evidence indicates that MSCs may be an ideal carrier for antitumor biologics such as cytokines (shown in Table 2), chemotherapeutic agents (shown in Table 3), and oncolytic viruses (shown in Table 4). The main reasons of which are listed as follows: (1) MSCs expressing transgenes maintain long-term expression in the body, because of their low immunogenic properties and the production of immunosuppressive molecules [86].  This cytokine has very strong antitumor and antiangiogenic properties [87]. Gene modification of MSCs by infection with an adenoviral or retroviral vector encoding human IL-12 augments the antitumor effect in melanoma, renal cell carcinoma, breast tumor, hepatoma, and glioma [51][52][53][54][55][56][57]. The antitumor effects of MSC-IL-12 depend on the stronger tumor-specific T-cell responses [51,57]. Moreover, the antitumor activity of the MSCs-IL-12 is related to the presence of natural killer (NK) cells and interferon-γ (IFN-γ) [52]. However, MSCs-IL-12 embedded in Matrigel exhibits significant antitumor effects even in immunodeficient mice lacking T, B, and NK cells, but not in IFN-γ knockout mice [51]. Fortunately, the intratumoral expression levels of IL-12 are enhanced by MSCs-IL-12 to be tenfold greater than that of free IL-12 groups in the ultimate stage [55]. A 20-day course of intravenous injection of MSC-IL-12 is without systemic toxic effects [55]. Similarly, the tumor growth is inhibited, and survival is prolonged in ovariancancer-bearing mice treated with MSCs-IL-21. The number of IFN-γ-secreting splenocytes and NK cytotoxicity significantly increase after MSCs-IL-21 administration [59].

Interferon (IFN). Type I interferons (IFN-α and -β)
show a variety of antitumor effects, including inhibiting cell proliferation, limiting tumor angiogenesis, inducing cell apoptosis, and activating the host's defense against tumors [88]. Systemic administration of MSCs-IFN-α significantly inhibits the growth of B16F10 melanoma cells and prolongs the survival. The result of immunohistochemical analysis reveals the promoted apoptosis and the decreased proliferation and vascular system [42]. IFN-β produced by MSCs inhibits the growth of malignant cells such as breast cancer, prostate cancer, bronchioloalveolar carcinoma, lung cancer, tongue squamous cell carcinoma, melanoma, lung metastatic melanoma, and pancreatic tumors metastases to the lung [31,[43][44][45][46][47][48]. Antitumor effect of IFN-β may be related to the increase of apoptosis [45] and NK cell activity [44] and 3 Stem Cells International  [43]. However, the combination of MSCs-IFNβ and anti-inflammatory drugs in the treatment of pancreatic tumors may lose these beneficial effects [31]. INF-β-MSCs specifically target tumor cells and do not cause damage to internal organs due to the use of INFβ alone [46]. The combination of canine MSCs-IFN-β and low-dose cisplatin improves the therapeutic effect of canine melanoma [48]. As a type II interferon, IFN-γ is mainly produced by lymphocytes and NK cells and plays an important role in the adaptive cellular immune response against tumors [88]. IFN-γ is considered to be a promising antitumor drug; however, the clinical application of the protein form of IFN-γ is hindered by serious side effects [89]. IFNγ-modified MSCs selectively induce lung tumor cell apoptosis through the activation of caspase-3 in vitro and inhibit the growth and progression of lung cancer in vivo [50].

Tumour Necrosis Factor-Related Apoptosis-Inducing
Ligand (TRAIL). TRAIL is a member of the tumor necrosis factor (TNF) cytokine superfamily with attractive potent antitumor function owing to its unique competence to target cancerous cells without any harm to adjacent normal cells. This is because the expression of TRAIL-specific receptors, termed death receptors, is significantly higher in cancer cells compared with normal cells [90].  [65]. However, BM-MSCs did not take up and release PMX in effective amounts on mesothelioma, although PTX-loaded BM-MSCs dramatically inhibited mesothelioma proliferation [68]. Therefore, we speculate that MSCs have a certain specificity for the absorption and release of drugs, which may be closely associated with the structure of the drug, the surface protein of the MSCs, and the tolerance of the MSCs to the drug. Considering current research primarily in vitro, whether the MSCs loaded with chemotherapeutic drugs are protected by the immune system in systematic administration remains to be explored. MSCs or engineered MSCs carried chemotherapeutic drugs may be a promising method for the treatment of drug-resistant tumors. The coculture of ovarian cancer cells with PTX-AT-MSCs inhibited cell viability in 2D and 3D models and counteracted PTX-resistance cells [64]. Coccè et al. demonstrated that AT-MSCs engineered with TRAIL were resistant to PTX and able to incorporate and then release the drug. The PTX delivery together with TRAIL secretion resulted in increased antitumor efficacy in human pancreatic carcinoma and glioblastoma in vitro [66].
The efficiency of drug-loaded nanoparticles is determined by the enhanced permeation and retention effect. As a result, the underperfused or hypoxic location within tumors rarely benefits from nanomedicine [71]. MSCs are recognized as ideal carriers of nanomedicine for tumortargeting therapy because of their tumor-homing potential in response to proinflammatory cytokines in the tumor microenvironment. MSCs are engineered with drug-loaded nanoparticles and result in an antitumor effect [71][72][73]. MSCs loaded with nano-PTX result in significant inhibition  [72]. A silica nanorattle-DOX drug delivery system is efficiently anchored to MSCs by specific antibody-antigen recognitions at the cytomembrane interface. MSCs-nano-DOX can track down the U251 glioma tumor cells and deliver DOX with wide distribution and long retention lifetime in tumor tissues with low systematic toxicity in vivo [73]. Zhao et al. [71] prepared DOX-loaded poly (d, l-lactic-co-glycolic acid) (PLGA) nanoparticles and loaded them in MSCs. The average DOX content was measured at 20:98 ± 4:02 pg/cell in PLGA-DOX-loaded MSCs, which resulted in improved drug concentration in the lungs and sites of the metastasis and enhanced antitumor efficacy [71].

MSCs as Delivery of Oncolytic
Virus. The oncolytic virus (OV) has shown promising results in various clinical trials for the treatment of various cancers. However, systemic administration of OV is severely restricted by their immunogenic nature and poor tumor-homing ability; thus, oncolytic adenovirus (OADV) cannot be utilized to treat disseminated metastases [74]. The MSC-based delivery system which could circumvent humoral immunity is an ideal solution. The success of this strategy depends on efficient ex vivo   Track metastatic tumor deposits in the brain, suppress brain tumor growth and prolong survival in mouse models of melanoma brain metastasis [82] Breast cancer metastases to the brain/ovarian cancer lung metastases Fetal membranederived MSCs In vivo OHSV i.v. Tumor growth inhibition of lung and brain metastases [83] Liver cancer BM-MSCs In vivo OMV i.v.
Inhibition of tumor growth in both measles antibody-naïve and passivelyimmunized SCID mice [84] Acute lymphoblastic leukemia BM-MSCs In vivo OMV i.v.
Inhibition of cancer development in a murine model of disseminated ALL following MSC-mediated delivery of OMV [85] MSC 7 Stem Cells International the antiapoptotic viral gene E1B19K or downregulated expression of the death ligand TRAIL increased virus titers released from MSCs, which resulted in improved killing of the pancreatic cancer cells [78]. Menstrual blood MSCs (MB-MSCs) combined with ICOVIR15-cBiTE, an OADV expressing an epidermal growth factor receptor-(EGFR-) targeting bispecific T-cell engager (cBiTE), enhanced the antitumor efficacy compared to MB-MSCs loaded with the unarmed virus ICOVIR15 [80]. Guo et al. demonstrated that MB-MSCs specifically targeted tumor cells and served as an OADV delivery platform. MB-MSCs loaded with OADV (CRAd5/F11) inhibited tumor growth in vivo and in vitro [76]. The growth of ovarian cancer lung metastases and breast cancer brain metastases were strongly inhibited after a single injection of MSCs loading OHSV retargeted to HER2 [83]. MSCs-OHSV resulted in significantly increased antiglioblastoma efficacy compared with direct injection of purified OHSV in a preclinical model of glioblastoma resection, ensuing in extended median survival in mice. And MSCs loaded with OHSV-TRAIL successfully induced apoptosis-mediated killing and extended median survival in mice bearing OHSV-and TRAIL-resistant glioblastoma in vitro [81].
Enhancement of antitumor efficacy of MSCs-OV not only benefits from its ability to circumvent humoral immunity to homing but also infiltration of immune cells and differentiation ability of MSCs. MSCs-OMV was protected from antiviral antibodies, which was why therapy with MSCs-OMV resulted in significant inhibition of tumor growth in both measles antibody-naïve and passively immunized SCID mice. By contrast, when OMV was delivered systemically alone, antitumor activity was evident only in measles antibody-naïve SCID mice [84]. Similarly, BM-MSCs enhanced the therapeutic efficacy of systemically delivered OMV in the presence of preexisting high titer anti-MV antibodies in the SCID murine model of acute lymphoblastic leukemia [85]. Rincón et al. suggested that the use of MSCs as carriers of OADV could improve the clinical efficacy of anticancer virotherapy, not only by driving the adenovirus to tumors but also through their potential to recruit T cells, including CD8+ and CD4+ T cells [77]. Intracarotiddelivered MSCs-OHSV, but not purified OHSV, efficiently tracked metastatic tumor deposits in the brain, suppressed brain tumor growth, and prolonged survival in mouse models of melanoma brain metastasis. Furthermore, an increased CD8 + IFNγ+ tumor-infiltrating T lymphocytes population was observed after injecting MSC-OHSV intracarotid [82]. Combination therapy of MSCs-OHSV and anti-PD-L1 improved therapeutic efficacy in a syngeneic mouse model of melanoma brain metastasis [82]. The UC-MSC-loaded OADV could be home to the tumor sites and differentiate into hepatocyte-like cells within the tumor microenvironment first. Subsequently, the OADV lysed tumor cells selectively to exhibit tumor inhibition on both orthotopic and subcutaneous hepatic xenograft tumor model mice with less toxicity on normal organs [79].

Effect of MSC Extracellular Vesicles (EVs)/ Exosomes on Tumors
Exosomes derived from MSCs serve as paracrine mediators to inhibit or promote tumor progression by transferring sig-naling molecules (shown in Table 5). Human UC-MSC-EVs exert potently antiproliferative and proapoptotic results on bladder tumor T24 cells both in vitro and in vivo through restraining phosphorylation of Akt and upregulating p-p53/p21 and cleaved Caspase 3 [92]. EVs derived from human BM-MSCs induce apoptosis to inhibit cell cycle progression and the growth of HepG2 hepatoma, Kaposi's sarcoma, and Skov-3 ovarian tumor cell lines in vitro and in vivo [93]. miR-16 shuttled by MSC-derived exosomes can downregulate the expression of VEGF in tumor cells to suppress angiogenesis and tumor progression in vitro and in vivo [95]. Normal BM-MSC exosomes miR-15a inhibit the growth of multiple myeloma cells, although multiple myeloma BM-MSC-derived exosomes promoted multiple myeloma tumor growth [94]. TRIM14 can promote the proliferation of AML cells via activating PI3K/AKT pathway, which is reversed by BM-MSC exosomes through delivering miR-23b-5p [100].
Nevertheless, it has also been reported that MSC-derived exosomes or EVs are involved in modulating tumor growth and advancing tumor progression. BM-MSC-derived EVs support the tumor growth and angiogenesis of breast cancer in vivo and in vitro [96]. BM-MSC-derived exosomes promote human osteosarcoma and gastric cancer cell proliferation through the activation of the hedgehog signaling pathway [97]. In addition, BM-MSC-derived exosomes enhance VEGF expression in tumor cells by activating the extracellular signal-regulated kinase1/2 (ERK1/2) pathway [98]. AT-MSC-derived exosomes promote migration of the breast cancer cell line MCF7 via activation of the Wnt signaling pathway [99].
It is worth noting that the effects of MSC exosomes or EVs from different sources on the same tumor cell line may be inconsistent. MSC exosomes derived from the same tissue may have different effects on different cell lines. For example, BM-MSC-derived exosomes inhibited the angiogenesis of the murine breast cancer cell line 4T1 [95], while AT-MSC-derived exosomes promote the migration of the human cell line MCF-7 [99]. In addition, BM-MSCderived EVs promote the proliferation and metastasis of human breast cancer cell line MCF-7 [96]. The effect of protumor or antitumor of MSC exosomes may be related to the source of MSCs, the protocol of MSC culture and exosome extraction, the heterogeneity of cancer cells, and the surrounding microenvironment. Given this, if natural MSC exosomes are used alone to treat tumors, the protocol of MSC culture and exosome extraction must be standardized, and the effects and mechanisms of MSC exosomes for specific tumors must be clarified. In addition, local treatment by intratumoral injection may be safer than systemic administration.

Extracellular Vesicles and Exosomes
Derived from MSCs as Antitumor Vectors The exosomes are smaller, less complex, and less immunogenic than their parent cells since they have a lower content of membrane-bound proteins [113]. In addition, exosomes contain transmembrane and membrane-anchored proteins 8 Stem Cells International that likely enhance endocytosis, thus promoting the delivery of their internal content [114]. As demonstrated by Smyth et al. [115], the internalization of exosomes within tumor cells is ten times greater than liposomes of comparable size, representing a superior specificity of exosomes for cancer targeting. EVs and exosomes derived from MSCs may be ideal antitumor vectors (shown in Table 6).
6.1. miRNA. miR-122-transfected AT-MSCs can efficaciously package miR-122 into secreted exosomes, which can mediate the miR-122 conversation between AT-MSCs and HCC cells, thereby rendering cancer cells sensitive to chemotherapeutic agents through alteration of miR-122target gene expression in HCC cells [106]. Systemic administration of miR-379-enriched EVs causes a significant reduction in tumor activity over the 6 weeks of monitoring [107]. Exosomal miR-9-3p secreted from BM-MSCs inhibits viability, migration, and invasion while promoting apoptosis in bladder cancer via the downregulation of ESM1 [112]. The delivery of miR-139-5p from UC-MSC-derived exosomes results in the repression of proliferation, invasion, and migration of T24 cells together with the inhibition of bladder tumorigenesis in nude mice [101]. BM-MSCderived exosomes expressing miR-146b inhibit glioma growth via decreasing expression of EGFR and NF-κB protein [104]. BM-MSC-derived exosomes containing miR-143 are easily transferred into recipient cells and suppressed the migration of osteosarcoma cell lines [105]. MSC exosomes are effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in  [108]. The delivery of anti-miR-9 to the resistant glioblastoma cells reverses the expression of the multidrug transporter and reverses the chemoresistance of glioblastoma cells [103].
6.2. Drugs. PTX-loaded MSC-derived EVs are loaded with PTX and possess strong antiproliferative activity on the pancreatic adenocarcinoma cell line CFPAC-1 [102]. PTXloaded MSC exosomes are successfully isolated using a  [110]. The MSC exosomes loaded with DOX are prepared by mixing exosome with DOX, desalinizing with triethylamine, and then dialyzing against PBS overnight. Compared with the free DOX, the prepared exosome-DOX shows enhanced cellular uptake efficiency and antitumor effect in the osteosarcoma MG63 cell line [111].

Conclusion and Prospect
Recently, it has been increasingly verified that MSCs play a prominent role in tumor growth, progression, and treatment response. However, the role of MSCs and EVs in tumor progression is controversial, with apparently contradictory results having been published. Such contradicting observations may be related to the heterogeneity of MSCs and experimental condition, such as the dose and time of MSC or EV administration and the optimal culture condition. Generation methods and culture systems of MSCs should be better standardized to make results more reproducible in future research. Furthermore, although MSCs are clinically used as therapeutic agents in inflammatory disease and regenerative medicine, the potential functional role of multipotential differentiation of MSCs during cancer development in vivo is so far uncertain. Large and stringently designed studies are required to answer whether MSCs contribute to carcinogenesis for people without tumors following systemic administration. Also, because of their tropism to the tumor and low immunogenic properties, MSCs have been recently developed as carriers for against cancer biologics like cytokines, chemotherapeutic agents, and OVs. Few studies focus on the biosafety of engineered MSCs, despite the fact that a small number of MSCs may be present in other regions of the body and may affect healthy tissues after delivery of modified MSCs. As a result, a comprehensive evaluation of the optimal dose, therapeutic routine, drug distribution, and biological safety of engineered MSCs in cancer treatment is required. Furthermore, most of the research examining the efficacy of engineered MSCs on tumors is mainly performed in cancer cells and/or animal models. It would be meaningful to launch clinical studies to demonstrate the safety and efficacy of engineered MSCs in tumors.
Herein, we present several promising research directions for the future. First, MSCs as vehicles of chemotherapeutic drugs augment the targeting delivery of antitumor efficacy to tumor sites and avoid toxicity and acquired resistance to chemotherapy. Next, whether MSC-loaded nanoparticles have an advantage over nanoparticles remains to be seen, such as reduction of nanotoxicity and circumventing immune rejection.

Conflicts of Interest
The authors declare that they have no competing interests.

Authors' Contributions
Enguang Yang and Suoshi Jing contributed equally to this work. Enguang Yang and Suoshi Jing drafted the manuscript. Yuhan Wang and Hanzhang Wang revised the manuscript. Zhiping Wang and Ronald Rodriguez conceived the hypothesis and contributed to the preparation of the final version of the manuscript. All authors reviewed the manuscript and approved the final manuscript.